Plant phenol as new hepatoprotective agents

ABSTRACT

This invention relates to the isolation of compound namely (−)-wikstromol and together with or associated with a therapeutically acceptable additive and usefull as hepatoprotective agent.

FIELD OF THE INVENTION

[0001] This invention relates to the isolation of compound namely (−)-wikstromol (4,4¹,8-trihydroxy-3, 3¹-dimethoxyliganan-9, 9¹-olide) from a plant source Cedrus deodara in significant yields. This invention also identifies the use of the compounds as a hepatoprotective agent.

PRIOR ART

[0002] The compound (−)-wikstromol as such do not find much commercial value. The structural formula of (−)-wikstromol is shown in FIG. 1. Lignans are widely distributed in angiosperms and gymnosperms. The range of their structures and biological activities is broad. (−)-wikstromol is found to be active against p-388 lymphocyte leukemia and anti-HIV activity (M. K. Khamlach, R. Dhal and E. Brown). [Premieres syntheses totales du (+)-wikstromol, da la (−)-Trachelogenine, De la (−)-Nortrachelogenine et des lignoides apparentes—Tetrahedran 48, 10115-10126 (1992)].

[0003] The wood of Cedrus deodara possesses diaphoretic, diuretic and carminative properties, and it is usefull in treatment of fevers, piles and pulmonary and urinary disorders. The extract of the bark is astringent and useful for fevers, diarrhoea and dysentery. The oleoresin of deodar and the dark-colored oil obtained from the wood are valued for their application for ulcers and skin diseases. [Ref: Wealth of India, Vol.II, P.108-10 (1950) (published by CSIR)].

[0004] A close look at literature suggests that the lignan, seco-isolariciresinalldeglycoside (SDG) which has been reported to possess multitude of activities is isolated from flaxseed [U.S. Pat. No. 5,846,944].

[0005] P. K. Agarwal and R. P. Rastogi (Phytochemistry vol 21, No 6, pp 1459-1461, 1982) reported isolation of two lignans meso-secoisolariciresinol and cedrusinin from Cedrus deodara.

[0006] The isolation of matariesional in 0.10% yield and (+)-wikstromol in 0.124% yield is reported from Wikstroemia viridiflora (Wikstromol, a new lignan from Wikstroemia viridizflora—Sheela Tandon and R. P. rastogi; Phytochemistry 1976, vol 15, pp 1789-1791).

[0007] The extraction of the lignans and other constituents namely (−)-nortrachelogenin, carinol and carissanol from Carissa edulis reported by Hans Achenbach, Reiner Waibel and Ivan Addae-Mensah, Ref. Phytochemistry, Vol.22, No.3, pp.749-753, 1983.

[0008] There is a considerable amount of epidemiological evidence indicating an association between diet rich in fruits and vegetables and a decreased risk of cardiovascular disease and certain forms of cancer. It is generally assumed that the active principles contributing to these protective effects are nothing but antioxidant phytochemicals.

[0009] Recent research is directed to find out phytochemicals from plant sources in highlighting the role of polyphenolic compounds of plant materials as antioxidants, antimutagenic, anti inflammatory, antiatherosclerotic, antidiabetic, antihepatotoxic and antimicrobial agents. [Overview of flax lignans by Neil D. Westcott and Alister D. Muir, Crop Utilization Section, Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107, Science Place, Saskatoon, SK S7N 0×2, Canada in Volumn II—January 2000—inform]. Alcoholic extract of stem C. deodara was found to have anti-cancer activity. [Ref Medicinal Plants of India (ICMR) Vol.I, 1976, pp.214 and 215].

[0010] Accordingly, the applicants conducted a detailed study on principles from Cedrus deodara and this investigation led to the isolation of active principles namely, wikstromol. These compounds although hitherto isolated from Wikstromia spp. were in low yields. Cedrus deodara hence is a new source for the lignans and its presence in this taxon in significantly high yields makes this invention more important.

OBJECTS OF THE INVENTION

[0011] The main object of the invention is to provide novel compositions containing (−)-wikstromol and useful as a hepatoprotective agent.

[0012] Yet another object of the invention is to provide methods for the isolation of (−)-wikstromol from Cedrus deodara.

SUMMARY OF THE INVENTION

[0013] Accordingly, the invention provides novel composition containing (−)-wikstromol and useful as hepatoprotective agent. The invention further provides method for the isolation of (−)-wikstromol from Cedrus deodara.

DETAILED DESCRIPTION

[0014] Accordingly, the invention provides a composition comprising an effective amount of (−)-wikstromol together with or associated with an additive and useful as a hepatoprotective agent.

[0015] In an embodiment, the additive is selected in such a manner that they do not affect or interfere with the efficacy of active principles of the composition. The additive is such that they enhance and do not retard the activity of the active ingredient i.e. (−)-wikstromol.

[0016] In another embodiment the additive is selected from nutrients such as carbohydrates, sugar, proteins and pharmaceutically acceptable carrier.

[0017] In still another embodiment the ratio of (−)-wikstromol with the additive is in the range between 0.4:10 to 2.0:10.

[0018] In yet another embodiment (−)-wikstromol present in an amount of 250-300 mg.

[0019] Further the invention provides a process for the isolation of (−)-wikstromol from the Cedrus deodara, said process comprising the steps of:

[0020] a) extraction of the pulverized plant parts of Cedrus deodara with solvents to remove the essential oils;

[0021] b) concentrating the extract under vacuum to obtain a residue;

[0022] c) adding ethyl acetate to the residue obtained in step (b);

[0023] d) separating the solvents by conventional methods;

[0024] e) subjecting the residue to a first elution with about 3% methanol in chloroform to obtain (−)-matairesinol; and

[0025] f) subjecting the residue of step (e) to a second elution with about 5% methanol to obtain (−)-wikstromol.

[0026] The solvents used in step (a) are hexane and chloroform.

[0027] In another embodiment, the plant parts of Cedrus deodara such as bark and leaves are used for extraction.

[0028] In yet another embodiment, the wasted plant parts of Cedrus deodara are employed for isolation of the said compound. Preferably, the waste left after extraction of essential oil from the plant parts is used in the process.

[0029] Accordingly, the compositions prepared can be used for the treatment as hepatoprotective agent, wherein an effective amount of (−)-wikstromol is administered to a subject in need thereof

[0030] (−)-Wikstromol may be administered together with or in combination with therapeutically acceptable additives. The effective amount of (−)-wikstromol that may be administered to a subject can be readily determined by a person skilled in the art. However, it is recommended that the dosage of (−)-wikstromol or administered may be in the range of 250 to 300 mg per dose, twice a day. While it is possible to administer the composition in routes as the oral route achieves desired, best results.

[0031] Compositions employing (−)-wikstromol may be prepared by conventional methods as may be known in the art. The compositions may be in the form of tablets, capsules or syrups, etc. suitable additives as may be known in the art may be selected for the preparation of these compositions.

[0032] In essence the focus of the invention is to provide method for using (−)-wikstromol for the preparation of compositions useful as a hepatoprotective agent.

[0033] The heartwood of Cedrus deodara finds extensive use in essential oil industry. The oil by name ‘cedar wood’ oil finds application in flavor and fragrances. The heartwood powder after extraction of essential oil is a by-product and waste. This invention relates to isolation and purification of the compound (−)-wikstromol and useful as hepatoprotective agents.

[0034] This present invention relates to the isolation of compound namely (−)-wikstromol (4,4¹,8-trihydroxy-3, 3¹-dimethoxyliganan-9, 9¹-olide) from a new plant Cedrus deodara. This invention also relates to new use of the compound as a hepatoprotective agent.

[0035] The present invention embodies isolation of (−)-wikstromol, antioxidant principle from an entirely new source and their antihepatotoxic property compared with known biologically proved anti hepatotoxic efficacies.

BRIEF DESCRIPTION OF DRAWINGS

[0036] The accompanying diagrams illustrate the invention wherein:

[0037]FIG. 1(a): represents hydroxyl substituted 3, 31 dimethoxylignan-9, 9¹-olide.

[0038]FIG. 1(b): represents (−)-wikstromol (4,4,8-trihydroxy-3, 3¹-dimethoxyliganan-9, 9¹-olide).

[0039]FIG. 2: is the graphical representation of the hepatoprotective activity of (−)-Wikstromol.

[0040] Some of the embodiments of the present invention are presented by the following examples, which should not be construed as limitations on the inventive scope of the invention.

EXAMPLES 1 Experimental Protocols a Process for the Isolation of Wikstromol

[0041] The dried wood powder of Cedrus deodara was loaded 200 g in a soxhlet apparatus. The powder was first extracted with hexane to remove the essential oil composition. The residue from the extraction of hexane was further extracted with chloroform. The chloroform extract was concentrated under vacuum. The thick syrupy residue was dissolved in ethyl acetate for about 50 g of residue around 60 ml of ethyl acetate. The isolation of residue in ethyl acetate was added drop wise to hexane 9 around 5 Lt. The solid separated 35-g was filtered off.

[0042] The solid was loaded on silica gel column 60 120 mesh, 3.5-cm dia. Column loaded to a height of 60 cm. Initially the column was eluted with chloroform followed by 3% methanol in chloroform of get Matairesinol.

[0043] Further elution of the column with 5% methanol in chloroform yielded (−)-Wikstromol.

[0044] The yield (−) wikstromol is around 8.0 g.

[0045] The compound (−)-wikstromol is obtained in 90% purity.

[0046] The spectrochemical and physical properties of (−)-wikstromol are as under:

[0047] (−)-Wikstromol

[0048] 1. Molecular formula: C₂₀H₂₂O₇

[0049] 2. ¹H-NMR: 2.40-2.55(2H, m), 2.65-2.80 (2H, m), 3.10-3.20(1H, d), 3.85(6H, d), 3.95(2H,br d) 5.60(2H, d), 6.50-6.80(6H, m)

[0050] 3. ¹³C-NMR: δ 31.50(C-7), 41.90(C-8), 43.74(C-7), 55.94(2 X -OMe), 70.26(O—CH₂—O), 76.33(—C—OH) 111.55, 112.81, 114.35, 114.56, 116.82, 121.42, 123.12, 126.20, 130.35, 144.27, 144.95, 146.59(12 X Ar—C)

[0051] 4. MS: 374(M⁺)

[0052] 5. [α]_(D): −30.90 (28° C.)

Example 2

[0053] Carbon tetrachloride is a classical method to induce free radical mediated hepatotoxicity. In order to evaluate the free radical mediated hepatotoxicity and heptoprotective properties in the compound (−)-wikstromol, this method was employed. Male wistar rats were selected for study. The compound was dissolved in a small amount of DMSO and reconstituted in gum acacia/water. The compound was given one hour before CCl₄ administration to the over-night fasted rats in the dose of 250 mg/kg-body weight orally. After one hour mixture of CCl₄ and liquid paraffin (1:1, v/v) in the dose of 2.5 ml/kg was given orally. Fasting was continued. However, water was provided ad-libitum. After Shrs of CCl₄ administration, blood was taken out from retroorbital plexus. Serum aspartase transaminase and serum alanine transaminase was measured as a marker of hepatotoxicity. Table 1 and FIG. 2 show significant protection offered by the compound, (−)-wikstromol. FIG. 2 shows that (−)-wikstromol significantly prevented CCl₄ induced toxicity in rats. TABLE 1 AST (U/L) ALT (U/L) Before After Before After CCl₄ control 233 ± 139 686 ± 142 37 ± 9 273 ± 80 (10) (10) (10) (10) (−)-Wikstromol 281 ± 91  557 ± 126 42 ± 7 188 ± 66 Treated + CCl₄  (5)  (5)  (5)  (5)

Example 3

[0054] Carbon tetrachloride induced toxicity was repeated as referred in the example 2 by changing the dosage level to 300 mg/kg-body weight orally. Fasting was continued. However, water was provided ad-libitum. After Shrs of CCI₄ administration, blood was taken out from retroorbital plexus. Serum aspartase transaminase and serum alanine transaminase was measured as a marker of hepatotoxicity. The (−)-wikstromol shows significant activity and prevented CCl₄ induced toxicity in rats.

[0055] In accordance with the practice of this invention, it has been found that (−)-wikstromol is isolated from a new plant source Cedrus deodara in significant yields. Also, it has been found that (−)-wikstromol show hepatoprotective properties.

Advantages

[0056] The compound (−)-wikstromol is used in pure form.

[0057] Hence, the usage is more advantageous than a mixture of compounds having similar properties, which are in current use. It is also important to note that the process of isolation of (−)-wikstromol is highly economical and can be used as hepatoprotective agent.

[0058] (−)-Wikstromol is used at a high degree of purity of over 90%. They are found to be highly effective when administered at a dosage of 250 to 300 mg/kg of body weight. 

1. A method for the prevention of hepatotoxicity comprising administering to a subject in need thereof a composition comprising (−)-wikstromol.
 2. The method of claim 1, wherein the composition further comprises an additive selected from the group consisting of a carbohydrate, a protein and a pharmaceutically acceptable carrier, and mixtures thereof.
 3. The method of claim 2, wherein the ratio of (−)-wikstromol to additive is in the range from 0.4:10 to 1:5.
 4. The method of claim 1 wherein the amount of (−)-wikstromol that is administered is in the range from 250 to 300 mg per dose.
 5. The method of claim 2 wherein the amount of (−)-wikstromol that is administered is in the range from 250 to 300 mg per dose.
 6. The method of claim 3 wherein the amount of (−)-wikstromol that is administered is in the range from 250 to 300 mg per dose.
 7. The method of claim 1, wherein the composition is administered twice per day.
 8. The method of claim 2, wherein the composition is administered twice per day.
 9. The method of claim 3, wherein the composition is administered twice per day.
 10. The method of claim 4, wherein the composition is administered twice per day.
 11. The method of claim 5, wherein the composition is administered twice per day.
 12. The method of claim 6, wherein the composition is administered twice per day.
 13. The method of claim 1, wherein the composition is administered orally.
 14. The method of claim 6, wherein the composition is administered orally.
 15. The method of claim 7, wherein the composition is administered orally.
 16. The method of claim 12, wherein the composition is administered orally. 